PROJECT SUMMARY Rapid evolutionary adaptation is the foundational process in biology and at the core of many problems facing humanity, including cancer, bacterial and viral diseases, evolution of drug and pesticide resistance, and biological response to global change. This project aims to build predictive understanding of rapid evolution in an outbred, sexual organism in natural habitats by generating large-scale datasets of genotypic and phenotypic evolution on seasonal timescales (~10-20 generations) of the genetic model Drosophila melanogaster. The project follows previous work in which it was established that D. melanogaster populations in several temperate populations on the East Coast of North America cycle phenotypically for a number of fitness-related traits and genotypically at hundreds of SNPs. This project aims to test whether these patterns reflect the action of strong, local directional selection or whether they are due primarily to biased sampling and recurrent migration from refugia. In order to distinguish between these two scenarios the project will use a three-pronged approach. First, large-scale field seasonal collections across 20+ locations in North America and Europe combined with deep pooled resequencing of the collections, phenotyping of a subset of flies, and environmental monitoring will provide a fine map of the tempo and mode of phenotypic and genotyping evolution across space and environmental variation. Second, the project will employ highly replicated field mesocosms in an experimental orchard to directly observe phenotypic and genotypic evolution of D. melanogaster in a way that eliminates migration as a possible source of evolution. The experimental mesocosms will be initiated from two large outbred populations initiated from either a set of ~150 inbred and sequenced strains that were or from ~10,000 inbred lines generated by this project. Phenotyping and pooled resequencing will allow fine-mapping of genotype to phenotype, genotype to fitness, and phenotype to fitness to the best extent currently feasible. Finally, the project will investigate the phenotypic effects of ~50 seasonal SNPs that cycle robustly in the field and in the experimental mesocosms and that match to the best extent possible the genomic and population genetic properties of seasonal SNPs overall. The phenotypic tests across a large panel of fitness-related phenotypes will be carried out using outbred, indoor cages that are fixed for the alternate states of the seasonal SNPs in question and using genomic editing using CRISPR/Cas9 technology in two common genetic backgrounds. Upon completion of the three aims the project will generate the most comprehensive analysis and data to date of genotypic and phenotypic rapid evolution in a natural environmental context for any outbred, sexual organism.